The complete mitochondrial genome of Stauroderus scalaris (Orthoptera: Acrididae) and its phylogenetic implication

Abstract

This study reports the complete mitochondrial genome (15,610 bp) of Stauroderus scalaris, a grasshopper species from Xinjiang, China, which comprises 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region. Phylogenetic analysis confirms its close evolutionary relationship with the genera Gomphocerus and Gomphocerippus, supporting its placement within the tribe Gomphocerini. These findings enrich the available mitochondrial genome resources for grasshoppers and provide valuable molecular data for subsequent research on the phylogeny and evolutionary history of the subfamily Gomphocerinae.

Introduction

According to the Orthoptera Species File (https://orthoptera.speciesfile.org/) (Cigliano et al. 2025), the genus Stauroderus Bolívar, 1897 belongs to the tribe Gomphocerini Fieber, 1853, within the subfamily Gomphocerinae Fieber, 1853. Currently, only three valid species are recognized worldwide. These include Stauroderus yunnaneus Uvarov, 1925, which is endemic to Yunnan Province, southwestern China; Stauroderus campestris Stål, 1861, mainly distributed in the coastal areas of southeastern Brazil; and Stauroderus scalaris Fischer von Waldheim, 1846 (Figure 1). Compared to the other two species, S. scalaris is a widely distributed Palearctic species (Zhao et al. 2012) spanning Europe, Central Asia, and northwest China. As a typical dominant species in high-altitude ecosystems, S. scalaris is widely distributed across meadow-steppe, subalpine meadows, forest belts within mountainous-steppe zones of Xinjiang Uygur Autonomous Region, predominantly at elevations between 1,200 and 2,400 m (Chen 1981a, 1981b). Although partial mitochondrial sequences (cytb and COII) have been reported for S. scalaris (Wang et al. 2012), complete mitogenomic data are still lacking for all extant species within the genus Stauroderus. Consequently, the first complete mitogenome of S. scalaris characterized in this study provides a crucial molecular basis for investigating the adaptive evolution and resolving phylogenetic relationships within the genus Stauroderus.

Figure 1.

Images of Stauroderus scalaris: (A) lateral view and (B) dorsal view. Both photographs were taken by Yuanjia Zhong.

Materials and methods

Experimental materials and sources

The adult specimen of S. scalaris was collected in July 2023 from Xinyuan County, Ili Kazakh Autonomous Prefecture, Xinjiang, China (43.334815 N, 83.984346 E), at an altitude of 1301.28 m. This specimen was individually preserved in a 10 mL centrifuge tube filled with sufficient anhydrous ethanol. Subsequently, the sample was transported to the laboratory and stored in a freezer at −20 °C in the International Center for the Collaborative Management of Cross-border Pests in Central Asia, China. The specimen was unambiguously identified as S. scalaris by integrating morphological diagnosis with molecular verification. Morphological identification was conducted following the taxonomic criteria outlined in Fauna Sinica: Insecta, Vol. 10 (Zheng and Xia 1998). Molecular verification was performed by comparing the newly generated 600 bp of cytb sequence against the reference sequence (GenBank accession no. JQ996610) in the GenBank database (https://www.ncbi.nlm.nih.gov/), yielding 100% sequence identity. It is registered under accession number XJNURS20230615 (Contact: Liang Yuan, yuanliang_314@163.com).

Mitochondrial genome assembly and annotation

After sampling the leg muscle tissue, the total genomic DNA was extracted using the Mag-MK Animal Genomic DNA Extraction Kit (Sangon Biotech). The DNA samples were sent to Beijing Biomarker Technologies Co., Ltd. for library construction and high-throughput sequencing on the Illumina NovaSeq6000 platform. After sequencing, a total of 3.48 gigabytes (GB) of raw data was obtained. Gene sequence assembly was performed using the idba_ud program within the IDBA software suite (Peng et al. 2012). Preliminary annotation was completed via the MitoZ (https://github.com/linzhi2013/MitoZ/wiki/Installation) website to generate gbf files (Meng et al. 2019). These gbf files were ultimately visualized as mitochondrial genome maps through the Proksee (https://proksee.ca/) (Grant et al. 2023). To evaluate sequencing depth, raw data were mapped back to the assembled genome using Bowtie2 (Langdon 2015). The alignment files were processed and sorted using Samtools, after which coverage metrics were calculated via awk. Finally, the sequencing depth distribution was visualized using the ggplot2 package in R, as presented in Fig. S1.

Phylogenetic analysis

To clarify the phylogenetic position of S. scalaris within Gomphocerinae, we reconstructed a phylogenetic tree using complete mitochondrial genomes from 24 representative orthopteran species available in GenBank. Acrida cinerea Thunberg, 1815 (Acrididae: Acridinae) was selected as the outgroup. The nucleotide sequences of the 13 protein-coding genes (PCGs) were extracted using PhyloSuite v1.2.3 (Zhang et al. 2020). For each species, the extracted PCG sequences were manually concatenated into a single continuous sequence. Multiple sequence alignment of these concatenated datasets was then performed using the MUSCLE algorithm implemented in MEGA11 (Tamura et al. 2021), yielding a final alignment matrix of 11,240 nucleotide positions. The optimal evolutionary model for the concatenated dataset was evaluated using MrModeltest v2.3 via the MrMTgui interface, which identified GTR+I + G as the best-fit model. Bayesian inference (BI) analysis was performed using MrBayes v3.1.2 under the Markov Chain Monte Carlo (MCMC) method. The MCMC algorithm was run for 1,000,000 generations, and the initial 25% of sampled trees were discarded as burn-in to estimate posterior probabilities. The resulting BI phylogenetic tree was edited and visualized using the iTOL online tool (https://itol.embl.de/) (Letunic and Bork 2024).

Results

Mitogenome organization

The circular mitogenome of S. scalaris has a length of 15,610 base pairs (bp), comprising 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and one control region (D-loop) (Figure 2). Sequencing depth analysis demonstrated continuous coverage across the entire mitochondrial genome with a high mean depth and no gaps, confirming the completeness of the assembly (Fig. S1).

Figure 2.

Mitochondrial map of Stauroderus scalaris. Different colors represent genes with different functions. Inner rings show base-composition metrics.

The overall nucleotide base composition consists of 42.57% A, 32.35% T, 14.25% G, and 10.81% C, featuring a slightly elevated A + T content (74.92%) and a relatively low G + C content (25.08%). The 13 PCGs have a cumulative length of 11,196 bp, accounting for 71.72% of the entire genome, with individual lengths ranging from 162 bp (ATP8) to 1,719 bp (ND5). All 13 PCGs utilize ATN as start codons. Except for CO3, ND5, and ND1, which terminate with the TAG stop codon, the remaining PCGs utilize the canonical TAA. The 22 tRNA genes have an aggregate length of 1,480 bp, with an A + T content of 72.44%. Furthermore, sequence analysis identified 8 gene overlaps (2–8 bp in length) and 21 intergenic spacers (1–26 bp in length) across the mitogenome.

Phylogenetic analysis

In this study, we integrated mitochondrial genomic data from 24 species and reconstructed the phylogeny of the subfamily Gomphocerinae. We performed Bayesian Inference (BI) analysis based on the concatenated nucleotide sequences of 13 protein-coding genes (PCGs), selecting Acrida cinerea as the outgroup. The resulting topology (Figure 3) exhibited high resolution, with most major nodes supported by maximal Bayesian posterior probabilities (PP = 1.00). S. scalaris was unambiguously positioned within the subfamily Gomphocerinae, forming a strongly supported monophyletic clade (PP = 1.00) with the genera Gomphocerus and Gomphocerippus. Specifically, S. scalaris was identified as the sister group to the clade comprising Gomphocerus licenti Chang, 1939, Gomphocerus sibiricus Linnaeus, 1767, and Gomphocerippus rufus Linnaeus, 1758.

Figure 3.

Phylogenetic relationships among 23 Gomphocerinae species were reconstructed based on the concatenated nucleotide sequences of 13 PCGs. Acrida cinerea was used as an outgroup. Numbers at the nodes indicate Bayesian posterior probabilities. Accession numbers are: G. licenti GQ180102 (Gao et al. 2009), G. sibiricus MK903592 (Chang et al. 2020), Go. rufus JX122541 (Chang et al. 2020), C. m. huabeiensis MK903587 (Chang et al. 2020), C. hebeiensis MH101364 unpublished, C. chayuensis PV216184 unpublished, C. qingzangensis NC072940 (Sun et al. 2023), C. huchengensis OQ2051971 (Sun et al. 2023), C. nemus MK903589 (Chang et al. 2020), C. albonemus OQ274913(Sun et al. 2023), C. hsiai OQ205191(Sun et al. 2023), C. dubius NC071964 unpublished, C. aethalinus MK903586(Chang et al. 2020), O. petraeus MK903596 (Chang et al. 2020), O. viridulus MN083203 (Chang et al. 2020), O. haemorrhoidalis MK903570 (Chang et al. 2020), A. f. fusca MH101367 unpublished, A. m. microptera OQ612708 unpublished, A. meridionalis NC039962 unpublished, A. coreana NC013805 unpublished, D. k. kraussi OQ470638 unpublished, D. tartarus OQ470639 unpublished, Acr. cinerea GU344100 unpublished.

Discussion and conclusion

This study reports for the first time the complete mitochondrial genome of S. scalaris from Xinjiang, China, enriching the currently limited mitochondrial genome resources of grasshoppers. Our substantial sequencing depth ensures high confidence in base-calling accuracy. The complete mitochondrial genome of S. scalaris was determined to be 15,610 bp in length. Its genomic structure and nucleotide composition are consistent with those of typical Acrididae mitogenomes (Huang et al. 2010; Zhao et al. 2018; Liu et al. 2025).

The phylogenetic reconstruction based on our mitochondrial dataset provided robust insights into the evolutionary position of S. scalaris within the subfamily Gomphocerinae. Our analysis strongly supports the monophyly of the tribe Gomphocerini, which includes the genera Gomphocerus, Gomphocerippus, Stauroderus, and Chorthippus Fieber, 1852, with maximal nodal support (PP = 1.00). Specifically, S. scalaris was firmly clustered as the sister group to the (Gomphocerus + Gomphocerippus) clade. In traditional taxonomy, Stauroderus exhibits pronounced morphological affinities with Gomphocerus and Gomphocerippus, especially in the morphology of the stridulatory pegs and wing venation (Harz and Kaltenbach 2013). Our molecular phylogeny is congruent with both traditional morphological systematics and recent molecular phylogenies (Wang et al. 2023), providing strong genetic evidence for their close relationship and common evolutionary origin. Furthermore, species within these genera are ecologically dominant in high-altitude regions; for example, both Gomphocerus sibiricus and Gomphocerippus rufus can inhabit elevations up to 2,500 m (Chen 1981b; Giuliano 2022). Consequently, their parallel evolution appears to have been shaped by shared adaptive pressures within similar ecological environments.

Insect mitogenomes are widely used in evolutionary studies due to their compact size, conserved gene arrangement, and rapid evolution. However, relying exclusively on them can obscure the true species history, as their strict maternal inheritance and lack of recombination often lead to incongruence with nuclear gene trees (Mi et al. 2025). Therefore, integrating nuclear markers in future studies is essential to comprehensively resolve the phylogeny of the subfamily Gomphocerinae.

Funding Statement

This study was supported by the 2024 Central Government Guided Local Science and Technology development Fund Project in Xinjiang Uygur Autonomous Region (ZYYD2024ZY01).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

This study does not involve any human, vertebrate or regulated invertebrates experiments and as such, ethical approval is not required. Samples were stored in alcohol-filled 10 mL centrifuge tubes in the field and laboratory to obtain fresh DNA. The sampling and researches conducted on Stauroderus scalaris are compliant with relevant regulations.

Data availability statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI under the accession no. PX872762. The associated BioProject, SRA, and BioSample numbers are PRJNA1401497, SRR36783428, and SAMN54565468, respectively.